Natural hexuronyl hexosaminoglycane sulfate, method for the preparation thereof and related therapeutical uses

ABSTRACT

Natural heteropolysaccharide, specifically hexuronyl hexosaminoglycane sulfate and, a process to obtain it, which substantially provides for the following steps: 
     (a) hydrolysis of an animal organ with a proteolytic enzyme, in water; 
     (b) precipitation of the hydrolyzed product with a water miscible solvent; 
     (c) solubilization of the precipitate in a solution of a salt of a strong mineral acid; 
     (d) addition of a quaternary ammonium halide to form a soluble complex and a precipitate which is separated; 
     (e) precipitation of such complex by dilution with water; 
     (f) isolation and solubilization of the precipitated complex in a salt solution according to step (c); 
     (g) precipitation of the final product with a water miscible solvent and drying thereof; 
     Such compound is utilized as active principle in pharmaceutical compositions, particularly suitable for the prevention of thrombotic states.

This is a division of application Ser. No. 930,540 filed Aug. 2, 1978.

The present invention relates to a natural heteropolysaccharide, moreparticularly to a natural hexuronyl hexosaminoglycane sulfate, asobtained by extraction of animal organs, which shows anticoagulant,antithrombotic and clearing activity.

Thrombosis often causes permanent invalidity and is one of the mostfrequent factors of death in the field of the cardiovascular diseases.

The generic term of thrombosis includes states of hypercoagulability,the origin of which is attributable to:

`Risk factors` which give place to a `thrombogenic state`, such as, forexample, smoke, stresses, the prolonged use of contraceptives ofprogestin type, etc.

Hereditary factors, such as the absence or the lack of inhibitors(particularly antithrombin III).

Etiologic factors having various and sometimes not yet well clarifiedorigin, such as modifications in platelet aggregation.

Factors related to a temporary slowing down of the blood circulation,such as takes place, for example, after surgical operations undernarcosis.

The pathological consequences resulting from a `thrombogenic` state asdetermined by one or more of the above listed factors can be thefollowing:

pulmonary, cerebral, coronaric thromboembolism and infarction.

thrombophlebitis, varicose syndromes.

intravascular disseminated coagulation.

In view of such relevant phenomena, to date recourse may be made to twotypes of strategy:

1. The use of thrombolytic agents.

2. The prevention of thrombogenic states and of their consequences.

Due to the gravity and high rate of possible progress of the thrombosis,it is evident that the second type of strategy would be far the best.

In order to deal with, on prevention basis, the problem of thethrombosis, two classes of drugs are available at present: the oralanticoagulants (coumarin and derivatives thereof) and heparin. The oralanticoagulants (coumarin and derivatives) act at the hepatic level byblocking two factors of the coagulation: proconvertin and prothrombin.These anticoagulants, however, are unsuitable for a prolonged therapy,and furthermore, show a poor antithrombotic activity since their actiondoes not concern other factors of the hemocoagulation particularlyinvolved in the genesis of the thrombosis, mainly the XA and theplatelet factors.

Under this point of view the heparin appears to be more advantageoussince it acts on several plasmatic factors of the hemocoagulation, andparticularly on the thrombin, on the XA factor and also on the XII, XIand IX factors, besides its action on the so called PF4 platelet factor.All these actions can be attributed to the specific capacity of theheparin of activating the inhibitor of the above listed coagulationfactors. This inhibitor, which is present in the plasma, is calledantithrombin III and requires the presence of heparin as co-factor fordeveloping its action.

Unfortunately the heparin therapy is objectionable for two reasons:first of all, it is active only by parental route and the effect thereofhas a duration not greater than 8 to 12 hours, whereby a time extendedprophylaxis, involving two daily injections of heparin, is difficult toachieve. Furthermore, the heparin has not only an antithrombotic effect,but also a total anticoagulant action. Now, although this second actionis sometimes favourable, in some cases the risk of hemorrhage, if thetherapy is not suited to the particular patient, can be a seriousproblem in front of the indisputable advantages of prophylaxis of thethrombosis.

It has been found and it is the main subject of the present invention,that a heteropolysaccharide, more particularly a hexuronylhexosaminoglycane sulfate, as obtained by extraction from animal organs,is not only endowed with anticoagulant, antithrombotic and clearingactivity, but can be administered both orally and parenterally, it canbe absorbed through the intestinal barrier and by topical way, and showsa ratio between antithrombotic activity and anticoagulant activity whichis favourable in comparison with heparin.

For the identification of the product of the present invention referenceis made to the following data:

Hexosamine after hydrolysis (reaction with p-dimethylaminobenzaldehyde):27±3%

Organic SO₄ ⁻⁻ after hydrolysis (titration with naphtarsone): 27±4%

Acetyl groups after hydrolysis: 7±1%

Sodium (atomic absorption): 10±2%

Molar ratio huronic acids: hexosamine:sulfate:acetyl: sodium=about1:1.2:2:1.2:3

Molecular weight range (chromatography by exclusion on gel):8.000-16.000

Specific rotation [α]_(D) ²⁰ =-15/-30°

Electrophoresis on cellulose acetate (buffer:pyridine acetic acidwater=1:10:229, pH 4.5 and development with toluidine blue):

One main band with electrophoretic mobility U=1.90-1.95×10⁻⁴ cm² v⁻¹s⁻¹.

I.R. spectrum: characteristic bands are observed at 1740, 1647, 1555,1375, 1235 and 1050 cm⁻¹.

Solubility: the heteropolysaccharide of the present invention is solublein water, in diluted mineral acids and diluted fixed alkalis butinsoluble in ethanol.

As regards the identification, the following test is furthermore useful:1 ml of a 2% water solution of the product of the present invention issupplemented with 3 mls of 2% water solution of cetylpyridinium chlorideheated to 40° C.: A bulky white precipitate is formed. The precipitateis separated by centrifugation and suspended in 3 mls of a 0.7 M KClsolution: the complete dissolution of the precipitate is obtained.

Another feature of the present invention relates to the extractionprocess for obtaining the above identified product. This process ischaracterized by the following steps:

(a) Hydrolysis of an animal organ, ground and suspended in water, with aproteolytic enzyme.

(b) Precipitation of the limpid liquid by a water miscible solvent.

(c) Solubilization of the precipitate in a solution of a salt of astrong mineral acid and of a mono- or divalent cation (e.g. sodium,ammonium, calcium) said saline solution having a ion concentrationcorresponding to that of 0.8 M NaCl.

(d) Addition at a temperature not higher than 80° C. of an excess ofquaternary ammonium halide, selected from those having in their moleculeat least one aliphatic group with more than 12 atoms, whereby a complexis formed which remains in solution, whereas the simultaneously formedprecipitate is separated.

(e) Precipitation of the complex by dilution with water until a ionconcentration not higher than that of 0.4 M NaCl is obtained.

(f) Isolation of the precipitated complex and solubilization thereof ina salt solution having the same characteristics of that of the step (c).

Precipitation of the desired product by a water miscible solvent anddesiccation.

By particularly considering now the several steps of the above definedprocess, the following features are to be pointed out. The animal organwhich is subjected to the hydrolysis is preferably fresh or deep-frozen.Amongst the useful animal organs, lungs and duodenum are preferred. Theproteolytic enzyme is preferably selected from vegetal (papain, ficin,bromelin) or bacterial endopeptidases, and the conditions in which thehydrolysis is carried out depends on the type of enzyme. It isparticularly preferred to perate the hydrolysis under mild heat, forperiods not less than 3 hours, and until the value of the α-aminonitrogen (as determined by means of the Soerensen Method) no longervaries.

For the precipitation of the limpid liquid after hydrolysis the solventto be used is selected between acetone, dioxane, methanol, ethanol andthe like.

The quaternary ammonium halide of the step (d) is preferably selectedfrom chlorides and bromides of cetylpyridinium andcetyltrimethylammonium. The related excess is of at least 0.5 g ofquaternary amonium salt per Kg of starting animal organ. Lastly for theprecipitation of the final product (step g) the use of acetone ispreferred and the precipitate is vacuum-dried or lyophilized.

There are now given two examples, having illustrative but not limitativepurpose, of the extraction process according to the present invention.

EXAMPLE 1

100 Kg of pork duodenum are ground and suspended into 50 liters ofwater. After heating to 40° C., 200 g of papain suspended into 25 litersof water are added. After heating the whole mixture to 60° C., the lysisis continued for 3 hours.

At the end, the mixture is heated to 90° C. for 15 minutes. The wholemass is filtered in a filterpress with 4 Kg of filter aid.

The limpid filtrate is concentrated to 50 liters, supplemented with 150liters of ethanol and maintained on standing overnight.

After removal of the hydro-alcoholic upper liquid phase, the precipitateis carefully dried and suspended into 25 liters of water containing 1200g of NaCl and filtered. The filtrate is supplemented under stirring with25 liters of 0.8 M NaCl containing 50 g of cetyltrimethylammoniumchloride and the mixture is heated to 40° C. The mixture is filtered ina filter-press with 2 Kg of filter aid and the limpid filtrate is addedwith 2 Kg of filter aid and diluted under stirring with 50 liters ofwater. After heating to 40° C., the precipitate is collected by vacuumfiltration.

The cake of precipitate is suspended into 10 liters of 0.8 M NaCl andheated to 40° C. After filtration, to the limpid liquid, under stirring,1.5 volumes of acetone are added, the mixture being maintained onstanding overnight. The hydroacetonic liquid upper phase is removed andthe precipitate is washed with 2×5 liters of 70% acetone and thendehydrated with anhydrous acetone. Then the pulverization and the vacuumdesiccation are carried out.

Yield: 8 g of white-ivory, sligthly hydroscopic powder, having thefollowing characteristics:

Solubility in water: Complete

[α]_(D) ²⁰ =-18°

Huronic acids: 28%

Hexosamines: 30%

Organic SO₄ ⁻⁻ : 26.5%

Anticoagulating activity: 25 U/mg (USP)

Antithrombotic/anticoagulant activity ratio (Yin's test/KCCT test)=2.

Clearing activity: 125 ILU/mg (international lipasemic units).

As regarding the clearing activity, it is related to the lipasemicactivity, namely to the minimum value of international lipasemic units(ILU) which are found in 1 liter of plasma, by administeringintravenously the substance to be tested to rats at the dose of 1 mg/Kgof body weight. In turn by international lipasemic unit, the lipasemicactivity is that which causes 1 micromole of oleic acid to be hydrolyzedeach minute.

EXAMPLE 2

50 Kg of bovine lung are ground and suspended into 75 liters of water.After heating to 40° C. 200 g of papain suspended into 25 liters ofwater are added and the mixture is heated to 65° C. for 3 hours. Afterthis time the mixture is heated to 90° C. for 30 minutes.

The liquid is then filtered and the filtrate is concentrated to 50liters and filtered again. The filtrate is supplemented with 100 litersof acetone and then maintained on standing overnight. After removal ofthe liquid upper phase, the precipitate is taken with 50 liters of 0.8 MNaCl containing 50 g of cetylpyridinium chloride. The mixture is thenheated to 40° C. and filtered with 2 Kg of Standard Supercell(filter-aid).

The filtrate is diluted with 50 liters of water containing 500 g ofStandard Supercell as a suspension. The precipitate cake is collected ona filter, suspended into 10 liters of 0.8 M NaCl and filtered. Thelimpid filtrate is washed by stirring with 2 liters of chloroform. Afterseparation of the chloroform phase, the water phase is filtered againand precipitated by means of 1.5 volumes of ethyl alcohol. Theprecipitate is then washed and dehydrated with alcohol, and thendissolved into 100 mls of water. The solution is filtered by means of amembrane and lyophilized.

Yield: 6.9 of white-ivory powder having the following characteristics:

Solubility in water: Complete

Rotation [α]_(D) ²⁰ =-24°

Huronic acids: 31.2%

Hexosamine: 33.1%

(organic) SO₄ ⁻⁻ : 22.8%

Anticlotting activity: 28 U/mg (USP)

Clearing activity: 115-ILU/mg

Antithrombotic/Anticlotting activity: 2.

The heteropolysaccharide of the present invention, as obtained by theprocess above has been tested to assess the pharmacological propertiesand activities thereof.

Acute Toxicology

When administered to guinea pigs, rats, mice and rabbits, no toxicsymptoms resulted up to a dose of 400 mg/Kg.

    ______________________________________                                        LD.sub.50                                                                             i.p.     (mouse)    1615     mg/Kg                                            oral     (mouse)    >6000    mg/Kg                                            i.v.     (mouse)    1000     mg/Kg                                    LD.sub.50                                                                             i.p.     (rat)      1463     mg/Kg                                            oral     (rat)      >6000    mg/Kg                                            i.v.     (rat)       150     mg/Kg                                    ______________________________________                                    

Clearing activity test

(1) Ediol Test: 100-50 ILU/mg

(2) Triton Test: serum lipid in triton-treated rats are significantlyreduced by administered HP-80.

(3) Atherogenic diet test: when added at the dose of 1% to asemipurified diet supplemented with cholesterol and cholic acid, HP-80is able to lower the serum lipids in rabbits.

In vitro anti-clotting and anti-thrombotic activities

Anticlotting activity (USP): 25-40 U/mg

Kaolin-Cephalin clotting time (KCCT): 14-22 U/mg

Yin's test/KCCT: 1.8-2.5 (Heparin=1)

Antithrombotic activity

A statistically significant effect on the movement of the thrombus isobtained, as determined by Chandler loop on rat plasma. In the light ofthe above activities a therapeutic use is foreseen, by oral andparenteral routes at daily dosages of 100 to 200 mg for the followingcases:

Prevention of post-operative thrombo-embolisms.

Prevention of thrombotic occurrences following a thrombohenic status,such as for example that occuring in fertile women subjected to aprolonged treatment with oral contraceptives.

Prevention of deep vein thrombosis.

Prevention of hypercoagulability states.

Correction of the hyperdislipidemic states (hyperdislipoproteinamia).

By percutaneous route, the following therapeutic uses are indicated:

Thrombophlebitis, phlebitis, contusions, and hematomas.

What we claim is:
 1. A process for obtaining hexuronyl hemoxaminoglycanesulfate, consisting essentially ofHexosamines: 29±3% Huronic acids:27±3% (organic) SO₄ ⁻⁻ :27±4% Acetyl groups: 7±1% Sodium: 10±2% suchthat the molar ration of buronic acids: hexosamine:sulfate:acetyl:sodium is 1:1.2:2:1.2:3 PM (chromatography by exclusionon gel): 8.000-16.000 Specific rotary power [α]_(D) ²⁰ =-30°/-15°Electrolhoresis on cellulose acetate (buffer:pyridine-acetic-acid-water=1:10:229, pH 4.5 and development withtoluidine blue): one main band with electrophoretic mobilityU=1.90-1.95×10⁻⁴ cm² v⁻¹ s⁻¹ I.R. spectrum: characteristic bands areobserved at 1740, 1647, 1555, 1375, 1235 and 1050 cm⁻¹ solubility:soluble in water, in diluted mineral acids and diluted fixed alkaliesbut insoluble in ethanol, comprising the steps of (a) hydrolyzing ananimal organ, which has been ground and suspended in water, with aproteolytic enzyme, precipitating the limpid liquid by a water misciblesolvent, (c) solubilizing the precipitate in a solution of a salt of astrong mineral acid with an alkali metal, alkaline-earth metal orammonium, said saline solution having a ionic power corresponding tothat of 0.8 M NaCl, (d) adding at a temperature not higher than 80° C.an excess of quaternary ammonium halide, selected from those having intheir molecule at least one aliphatic group containing more than 12carbon atoms, whereby a complex is formed which remains in solution, andseparating the simultaneously formed precipitate, (e) precipitating thecomplex by dilution with water until an ionic power not higher than thatof 0.4 M NaCl is obtained, (f) isolating the precipitated complex andsolubilizing said complex in a salt solution having the samecharacteristics of that of step (c), and (g) precipitating of thehexuronyl hexosaminoglycane sulfate with a water miscible solvent anddrying thereof.
 2. A process according to claim 1 wherein the tissueemployed is lung tissue.
 3. A process as claimed in claim 1,characterized in that the hydrolysis with proteolytic enzyme is carriedout on lung or duodenum tissue.
 4. A process as claimed in either one ofclaim 1 or claim 3, characterized in that the proteolytic enzyme isselected from papain, bromelin, ficin, and bacterial proteolytic enzyme.5. A process as claimed in claim 1, characterized in that the enzymatichydrolysis is carried out under heat and until the value of the α-aminicnitrogen no longer varies.
 6. A process as claimed in claim 1,characterized in that the hydrolysis with the proteolytic enzyme iscontinued for at least 3 hours.
 7. A process as claimed in claim 1,characterized in that the said water-miscible solvent of the hydrolysislimpid liquid is selected from acetone, dioxane, methanol and ethanol.8. A process as claimed in claim 1, characterized in that the saidquaternary ammonium halide is selected from chlorides and bromides ofcetylpyridinium and cetyltrimethylammonium.
 9. A process as claimed inclaim 1, characterized in that the excess of quaternary ammonium halideis at least 0.5 g per Kg of the starting organ.
 10. A process as claimedin claim 1, characterized in that the said water-miscible solvent in thefinal precipitation step (step g) is acetone.